We have characterized the reactive intermediates of the peroxidase-like reaction of Bulkholderia pseudomallei KatG using multifrequency EPR spectroscopy. The aim was to investigate the putative role of tryptophanyl radicals as alternative intermediates to the [Fe(IV)=O Por(center dot+)] species or as short-lived species involved in superexchange-coupled pathways between redox cofactors. Three distinct sites for the formation of radical intermediates, Trp330, Trpl 39 and Trpl 53, were identified using single, double and triple variants of Bulkholderia pseudomallei KatG. The proximal Trp330 is the site for a radical in magnetic interaction with the ferryl heme iron [Fe(IV)=O Trp(center dot+)], formed at the expense of a short-lived (Fe(IV)=O Por(center dot+)] species as in the cases of Mycobacterium tuberculosis KatG and cytochrome c peroxidase. Formation of the Trpl 53 radical at a site close to the enzyme surface crucially depends on the integrity of the H-bonding network of the heme distal side, that includes Trp95, the radical site in the Synechocystis KatG. Accordingly, the extended H-bonding network and Trp94 provide an electron transfer pathway between Trp153 and the heme. The distal tryptophan (Trp 111) being part of the KatG-specific adduct required for the catalase-like activity, is involved in facilitating electron transfer for the formation of the Trpl 39 radical. We propose a comprehensive description of the role of specific Trip residues that takes into account not only the apparent differences in sites for the Trp(center dot) intermediates in other catalase-peroxidases but also the similar cases observed in monofunctional peroxidases.